PROJECT SUMMARY Multiphoton microscopy (MPM) can provide sub-micron resolution images of living tissues in their native environment with label-free molecular contrast from multiple modalities, including second harmonic generation (SHG) and two-photon excited fluorescence (TPEF). Several endogenous tissue components can be visualized, including collagen (from SHG) and reduced nicotinamide adenine dinucleotide (NADH), flavin adenosine dinucleotide (FAD), keratin, melanin and elastin fibers (from TPEF). We have advanced label-free MPM technologies in skin clinical/translational studies for characterizing keratinocyte metabolism, diagnosing melanoma, understanding melanocyte biology, detecting basal cell carcinoma, quantifying skin pigmentation, and assessing the effects of cutaneous laser therapy. Many of these studies have been completed using a commercial multi-photon microscope for clinical skin imaging that has limitations in terms of field-of-view (FOV), speed, footprint, and cost. In order to address these barriers to clinical adoption, we propose to build a ?next-generation? clinical multiphoton microscope that integrates advanced benchtop technologies into a compact, practical, and cost-effective bedside device. This new instrument will have comparable FOV, resolution, and scanning features to standard-of-care reflectance confocal microscopes (RCM), yet provide unique structural and metabolic contrast from multiple modalities (TPEF and SHG) that can only be achieved with MPM. We will establish the clinical safety of this device in a light dose escalation study that assesses DNA and cellular damage, and establish key performance benchmarks in a 12-patient clinical study of healthy volunteers across a range of skin types. In addition, we will conduct pilot studies of wound re-epithelialization and melanocyte migration in the context of vitiligo micro-grafts, a clinical procedure where pigmented skin is transplanted into skin affected by vitiligo, which is devoid of melanocytes. Melanocytes migrating out of engrafted skin and keratinocytes turning over within engrafted skin can be visualized by measuring the TPEF of cellular melanin and co-factors (NADH, FAD+) in and around the grafts, effectively identifying different cell populations involved in wound healing. Our broad, long term goal is to develop ev-MPM as a practical approach for rapid, in vivo characterization of cellular morphologic and metabolic imaging endpoints in patients. These can be used to understand and optimize wound healing and provide a practical beside platform for detecting, diagnosing, and optimizing therapeutic response in skin diseases.